No band after digestion - (Aug/11/2010 )

I was wondering if anyone could help me. I have tried to clone 240 bp PCR product cutting with NdeI/XhoI (restriction site in the primer) into pET23a+ cut with the same enzyme for a long time. There was no positive clone after many times cloning. After changing the host strain to K12, there were clones and then I extracted the plasmid with 5 PRIME kit and double digested with NdeI/XhoI. After running the gel, plasmid band was found but no or empty band in the digested plasmid. The control pET23a cut with NdeI/XhoI showed correct band. These showed no error in water, RE , buffer or BSA). Please help me to clarify this.

Thanks in advance

-Pig-

Hello

How are you purifying your PCR product?

Clare

Pig on Wed Aug 11 09:30:22 2010 said:

Hi,

I was wondering if anyone could help me. I have tried to clone 240 bp PCR product cutting with NdeI/XhoI (restriction site in the primer) into pET23a+ cut with the same enzyme for a long time. There was no positive clone after many times cloning. After changing the host strain to K12, there were clones and then I extracted the plasmid with 5 PRIME kit and double digested with NdeI/XhoI. After running the gel, plasmid band was found but no or empty band in the digested plasmid. The control pET23a cut with NdeI/XhoI showed correct band. These showed no error in water, RE , buffer or BSA). Please help me to clarify this.

Thanks in advance

-Clare-

When you prep the plasmid did you make sure the ethanol was added in PE ?